This invention is directed to an automated Total Dietary Fiber (TDF) analysis system which exhibits improved efficiency in filtration, labor and time, and eliminates the glassware and the associated glassware cleanup.
A variety of methods have been developed for the analysis of fiber in feeds and foods. Generally accepted methods for analyzing feeds in animal nutrition are Crude Fiber (AOAC Method 962.09) and Neutral Detergent Fiber and Acid Detergent Fiber (USDA, Agricultural Handbook No. 379). They are all gravimetric procedures and rely on filtration to isolate the fiber fraction. New fiber methods (Crude Fiber Analysis, AOCS method Ba 6a-05 and the ANKOM patent U.S. Pat. No. 5,370,007) have been developed that use filter bags to improve the filtration step and enable batch processing. AOAC refers to Association of Official Analytical Chemists and AOCS refers to American Oil Chemists Society. In human nutrition Total Dietary Fiber (TDF) is the term used to classify fiber components that have certain nutritional and digestive tract benefits. Considerably different from the animal fiber methods, the TDF methods have an additional requirement to precipitate the water soluble fiber fraction using alcohol. Thus fiber components that are water soluble in the early enzymatic stages of the method are later precipitated with alcohol and recovered as a part of the fiber fraction.
AOAC Official Method 991.43 (one of the approved TDF methods) first performs an enzymatic digestion of the starch and protein in the sample by treating it in a buffer solution with alpha amylase, then with protease, and finally, after the appropriate pH adjustment, with amyloglucosidase (AMG). TDF consists of two components; an insoluble dietary fiber (IDF) fraction and a soluble dietary fiber (SDF) fraction. TDF can be determined either by filtering the IDF and SDF fractions together in one filter, or by filtering the IDF and SDF fractions separately and then adding the two values together. In order to analyze the IDF and SDF fractions separately, the IDF fraction is filtered at the end of the enzyme digestion phase. Four volumes of ethanol are added to the filtrate to precipitate the SDF fraction. The subsequent precipitant is then separated by filtration. These filtrations are commonly time consuming and difficult. The process requires a fritted glass crucible with a layer of diatomaceous earth and a vacuum system to draw the liquid through the filter. In many samples the IDF and the SDF tend to coat the filter and inhibit the liquid passage, requiring extended periods of filtration. To facilitate filtration, the surface of the diatomaceous earth filter pad often requires scraping.
Transferring the entire sample quantitatively at two different times during the analysis (once to the IDF filter and once to the SDF filter) is critical. A fine precipitation of the SDF fraction coats the beaker walls and requires physical removal. The technician must take great care to scrape and rinse the beaker walls in order to transfer all of the fiber into the filtering crucible. Both the IDF and the SDF fractions are recovered in the filtering crucible. The quantities are determined gravimetrically by drying the crucible, weighing and subtracting the weight of the crucible (along with the diatomaceous earth filter pad), and correcting the sample for ash and protein. Those skilled in the art will understand that correcting the sample for ash involves burning a duplicate sample and measuring the remaining ash. Correcting for protein involves analyzing a duplicate sample using the Kjeldahl or Dumas method to measure the amount of protein in the sample. The IDF and SDF weight values are adjusted based on the ash and protein values. Total Dietary Fiber can be calculated by adding the IDF and SDF values after they have been corrected for ash and protein.
As can be seen from the above description, the analysis of dietary fiber is a long and arduous procedure with problematic transfer and filtration steps. Accurate control of conditions and careful quantitative transfers are required by the technician to produce accurate and precise results. Every particle in the digestion flask must be transferred to the filtering crucible and the diatomaceous earth filter pad must remain intact during the transfer in order to effectively capture the fine particulate. Filtration for many samples is slow even with strong vacuum assistance. Some samples may require a scraping of the filter pad to renew some of the filter surface in order to complete the filtration process. The scraping process must be accomplished without exposing the course filtering frit of the crucible. Many of the precision problems associated with this method are related to the difficulty of the transfer and filter processes.